Anti-cancer compositions containing Wnt decoy receptor

ABSTRACT

The present invention relates to a composition for preventing or treating cancer comprising Wnt decoy receptor. The composition of the present invention or the expression product thereof inhibits cancer generation, growth, proliferation and metastasis, and induces apoptosis of cancer cells, by binding to Wnt ligand and blocking ligand-receptor interactions, therefore may be effectively used as an anti-cancer agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.14/239,259, filed 4 Mar. 2014, which is a national phase application ofPCT Application No. PCT/KR2012/006527, filed on 16 Aug. 2012, whichclaims benefit of Korean Patent Application 10-2011-0082087, filed on 18Aug. 2011. The entire disclosure of the applications identified in thisparagraph is incorporated herein by references

FIELD

The present invention relates to an anti-cancer composition containingWnt decoy receptor which inhibits activation of Wnt signaling involvedin tumor development.

BACKGROUND

Lung cancer is highly aggressive and the most common cause ofcancer-related deaths worldwide. In 2009, the American Cancer Societyestimated that there were 219,440 new cases of lung cancer in the UnitedStates. Standard therapies such as surgery and radiation are noteffective in many cases (1); however, an increased understanding of themolecular mechanisms of lung cancer has led to the development ofpromising new therapies (2). Although chemotherapy advances haveimproved overall survival for patients with aggressive non-small celllung cancer, chemo resistance remains a major cause of treatment failure(3). Activating mutations in the epithelial growth factor receptor(EGFR) are present in a subset of lung adenocarcinomas, making thesetumors highly resistant to EGFR tyrosine kinase inhibitors gefitinib anderlotinib (4, 5). Many aggressive lung cancers show alterations invarious cancer-associated genes, including Wnt, K-ras, extracellularsignal-regulated kinase (ERK), Akt, and cyclooxygenase-2, suggesting adifferent molecular pathway for carcinogenesis in lung adenocarcinomas(6-8).

The role of Wnt signaling in cancer was first suggested 20 years agowith the discovery of Wnt-1 as an integration site for mouse mammarytumor virus (9). Many studies have reported that altered expression ofWnt ligands, receptors, and extracellular antagonists are associatedwith cancer development/progression and stem cellself-renewal/differentiation (10). Expression of the Wnt ligand,low-density lipoprotein receptor-related protein 5 (LRP5), and LRP6 areupregulated in lung cancers, whereas Wnt antagonists that bind Wntligands to block interaction with receptors (e.g., Wnt inhibitoryfactor-1 (WIF-1), secreted Frizzled-related proteins (sFRP) and dickkopfproteins (DKK) are downregulated or inactivated (11, 12). Accordingly,monoclonal antibodies and small interfering RNAs against Wnt andoverexpression of Wnt antagonists suppress tumor growth in various invitro and in vivo tumor models.

LRP6, a member of the LRP superfamily, is required for activation of thecanonical Wnt signaling pathway, which leads to the stabilization andnuclear translocation of β-catenin, the key effector molecule (13). LRP6consists of four distinct YWTD β-propeller/EGF-like domain pairs; thefirst and second YWTD domains (E1 and E2) are required for binding toWnt (14-16). In the present study, we explored the therapeutic utilityof a novel soluble Wnt receptor, sLRP6E1E2, which is composed of theLRP6 E1 and E2 regions. We examined the biological effects of sLRP6E1E2binding to extracellular Wnt ligands and blocking ligand-receptorinteractions. The results of the present invention provide directevidence that specific Wnt ligand/receptor interactions have potentialuse as anticancer therapeutic agents.

Throughout this application, various publications and patents arereferred and citations are provided in parentheses. The disclosures ofthese publications and patents in their entities are hereby incorporatedby references into this application in order to fully describe thisinvention and the state of the art to which this invention pertains.

SUMMARY

The present inventors have made intensive studies to develop a novelcomposition for cancer gene therapy with maximized inhibitory activityagainst cancer development/progression. As results, the presentinventors have discovered that novel soluble Wnt receptor, sLRP6E1E2,inhibits activation of Wnt signaling pathway in a cancer cell throughbinding to Wnt 3a protein, thereby reducing tumor growth, proliferationand metastasis and inducing apoptosis of tumor cells.

Accordingly, it is an object of this invention to provide a compositionfor preventing or treating cancer.

It is another object of this invention to provide a method forpreventing or treating cancer.

Other objects and advantages of the present invention will becomeapparent from the following detailed description together with theappended claims and drawings.

DRAWINGS

FIGS. 1A, 1B, 1C, and 1D represent characterization of the decoy Wntreceptor sLRP6E1E2. FIG. 1A is schematic representation of the genomicstructure of Ad vectors used. FIG. 1B shows endogenous Wnt3a expressionin several human lung cancer cell lines. FIG. 1C shows secretion andexpression of sLRP6E1E2. Cell culture supernatants were assessed withFLAG- or LRP6-specific Ab. (d) H322 and H460 cells were transduced withdE1-k35/LacZ or dE1-k35/sLRP6E1E2 (50 MOI) for 48 hr. Cell lysates wereimmunoprecipitated with antisera against Wnt3a (IP: Wnt3a) or LRP6 (IP:LRP6) followed by western blot (WB) analysis with the same antibodies.

FIGS. 2A, 2B, and 2C show that decoy Wnt receptor sLRP6E1E2 reducescytosolic β-catenin and T-cell factor transcriptional activity. FIG. 2Arepresents the result of TCF/LEF luciferase reporter assay in A549cells. *P<0.05 versus dE1-k35/LacZ-transduced or PBS-treated cells. FIG.2B represents the result of TCF/LEF luciferase reporter assay in H460and H322 cells. *P<0.05 versus PBS or dE1-k35/LacZ-transduced cells withor without Wnt3a. FIG. 2C shows H322 cells transduced with dE1-k35/LacZor dE1-k35/sLRP6E1E2 (50 MOI) with or without Wnt3a and labeled withanti-β-catenin. Original magnification, ×630.

FIGS. 3A, 3B, and 3C show that decoy Wnt receptor sLRP6E1E2 decreasesproliferation in human lung cancer cells. FIG. 3A shows A549 and H322cells transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (20 MOI). Thenext day, these cells were incubated with or without Wnt3a (100 ng/ml).After 3 days, cell proliferation was assessed by the MTT assay(mean±SEM). *P<0.05, #P<0.01 versus untreated control for each group;**P<0.001 versus dE1-k35/LacZ-transduced or PBS-treated cells. n.s.=notsignificant. FIG. 3B represents the result of western blot analysis forA549 cells treated as indicated above (FIG. 3A) using antibodiesspecific to Dvl2, Axin, Cyclin D1, or GSK-3β. FIG. 3C represents theresult of western blot analysis for MEK, ERK, Survivin, mTOR, PI3K, andAkt in H460 cells treated 50 MOI as indicated above (FIG. 3A).

FIGS. 4A, 4B, 4C, 4D, 4E, and 4F show that decoy Wnt receptor sLRP6E1E2induces apoptosis in human lung cancer cells. FIG. 4A shows photographstaken 72 hr after cells were transduced with dE1-k35/LacZ ordE1-k35/sLRP6E1E2 at (20 MOI). Original magnification, ×200. FIG. 4Brepresents detection of sLRP6E1E2-induced apoptosis by TUNEL staining.Original magnification, ×400. FIG. 4C indicates total number ofTUNEL-positive cells per fields (mean±SEM). *P<0.05 versus PBS ordE1-k35/LacZ treated with Wnt3a; **P<0.001 versus PBS-treated ordE1-k35/LacZ-transduced controls. n.s.=not significant. FIG. 4Drepresents the result of western blot analysis of sLRP6E1E2-mediatedapoptosis. H460 cells were transduced with dE1-k35/LacZ ordE1-k35/sLRP6E1E2 (20 MOI). The western blot using specific antibodiesagainst uncleaved PARP, cleaved PARP, pro-caspase-3, cleaved caspase-3,and cytochrome c. FIG. 4E represents subcellular localization ofcytochrome c in H460 cells treated as indicated above (FIG. 4D),determined by western blot analysis of cytosolic and microsomalfractions. FIG. 4F shows represents laser fluorescence confocalmicroscopy image of A549 cells treated as indicated above (FIG. 4D).Cells were stained with anti-cytochrome c (green) and MitoTracker (red).

FIGS. 5A, 5B, 5C, 5D, 5E, and 5F show that decoy Wnt receptor sLRP6E1E2inhibits tumor growth and characterization. FIG. 5A shows the tumorvolume on days 1, 3, and 5 after tumors were injected with PBS (▪),dE1-k35/LacZ (♦), RdB-k35 (▴), dE1-k35/sLRP6E1E2 (x), orRdB-k35/sLRP6E1E2 (●). Results are expressed as mean±SEM (n=7). *P<0.05versus PBS-treated or dE1-k35-treated controls and versusdE1-k35/sLRP6E1E2. #P<0.01 versus PBS-treated or dE1-k35-treatedcontrols. FIG. 5B shows tumor sections from each group wereimmunostained against E1A or FLAG (original magnification, ×40 and×100). FIG. 5C shows images of tumor tissues from each group stainedwith DAPI (blue), anti-Ki67 (red), and TdT-mediated TUNEL (green).Original magnification: ×100. FIG. 5D shows blood vessels visualized bystaining for CD31. Original magnification, ×100. FIG. 5E indicates meanmicrovessel density for each treatment group (CD31 positivecells/field). Results are expressed as mean±SEM (each group, n=3tumors). *P<0.05 versus PBS, dE1-k35, or dE1-k35/sLRP6E1E2. n.s.=notsignificant. FIG. 5F shows images of cells stained with DAPI (blue),anti-Wnt3a (red), or anti-β-catenin (green). Original magnification:×100.

FIGS. 6A, 6B, and 6C show the disruption of cell-cell junctions andepithelial-to-mesenchymal transition in tumor cells by Wnt3a treatment.FIG. 6A represents morphology changes of H322 cells by treatment ofWnt3a (100 ng/ml) for the indicated times. Original magnification, ×200.FIG. 6B shows E-cadherin, Vimentin, and β-catenin mRNA levels in H322cells after Wnt3a treatment. FIG. 6C shows images of H322 cells stainedwith DAPI (blue), TRITC-labeled phalloidin (red), or anti E-cadherin(green) after 24 incubation with or without Wnt3a (100 ng/ml). Originalmagnification, ×630.

FIGS. 7A, 7B, 7C, and 7D show that Decoy Wnt receptor sLRP6E1E2 inhibitscancer cell migration and invasion, and modulates expression ofepithelial-to-mesenchymal transition markers and MMPs. FIG. 7Arepresents the result of quantitative analysis of A549 lung cancer cellmigration. Experiments were performed in triplicate, and results areexpressed as mean±SEM. *P<0.05 versus PBS or dE1-k35/LacZ treatedcontrols; **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a. FIG. 7Brepresents invasion of tumor cells quantified as number of cells in fivefields of view per filter. Experiments were performed in triplicate, andresults are expressed as mean±SEM. *P<0.05 versus PBS or dE1-k35/LacZtreated controls; **P<0.001 versus PBS or dE1-k35/LacZ with Wnt3a. FIG.7C shows expression of EMT markers in H322 cells after 24 hr treatmentwith PBS, dE1-k35/LacZ, or dE1-k35/sLRP6E1E2 in the presence and absenceof Wnt3a (100 ng/ml). Cells were stained with DAPI (blue), TRITC-labeledphalloidin (red), or anti E-cadherin (green). Original magnification,×630. FIG. 7D shows expression of MMP-2 and MMP-9 mRNA levels in A549cells treated as indicated above (FIG. 3A), analyzed by semiquantitativeRT-PCR.

DETAILED DESCRIPTION

In one aspect of this invention, there is provided a composition forpreventing or treating cancer, comprising a polypeptide having the aminoacid sequence of SEQ ID NO:2.

In another aspect of this invention, there is provided a method forpreventing or treating cancer comprising administering to a subject inneed thereof a pharmaceutically effective amount of a polypeptide havingthe amino acid sequence of SEQ ID NO:2.

The present inventors have made intensive studies to develop a novelcomposition for cancer gene therapy with maximized inhibitory activityagainst cancer development/progression. As results, the presentinventors have discovered that novel soluble Wnt receptor, sLRP6E1E2,inhibits activation of Wnt signaling pathway in a cancer cell throughbinding to Wnt 3a protein, thereby reducing tumor growth, proliferationand metastasis and inducing apoptosis of tumor cells.

The term “polypeptide” as used herein, refers to a linear moleculeformed by peptide bonds between amino acid residues. The SEQ ID NO:2 isthe amino acid sequence of sLRP6E1E2 which is composed of the LRP6 E1and E2 regions. According to the present invention, sLRP6E1E2 inhibitsactivation of Wnt signaling pathway which plays a critical role incancer generation, growth, proliferation and metastasis, by blockingligand-receptor interactions through binding to Wnt ligand.

More concretely, soluble Wnt receptor of the present invention reducescytosolic β-catenin level and TCF transcriptional activity, inhibitscancer cell proliferation, induces cancer cell apoptosis, inhibits tumorgrowth, reduces expression of MMP-2 and MMP-9, which play a criticalrole in angiogenesis, tumor growth, and metastasis. Therefore, thecomposition of the present invention may be used as an anti-cancer agenteffectively suppressing cancer progression

The sLRP6E1E2 polypeptide of this invention may encompass sequenceshaving substantial identity to the amino acid sequence of SEQ ID NO:2.Sequences having the substantial identity show at least 80%, morepreferably at least 90%, most preferably at least 95% similarity to theamino acid sequence of sLRP6E1E2, as measured using one of the commonlyused sequence comparison algorithms.

In addition, the sLRP6E1E2 polypeptide of this invention includes theprotein having variant amino acid sequence as well as natural-occurringone. The variant of sLRP6E1E2 polypeptide refers to a protein ofdifferent sequence with deletion, insertion, conservative ornon-conservative substitution or combination thereof in one or moreamino acid residues. Such alteration of amino acid residues not tosubstantially impair protein activity is well known to one skilled inthe art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York,1979). Most common amino acid alteration includes Ala/Ser, Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, butnot limited to.

Optionally, the Nkx3.2 protein may be modified by phosphorylation,sulfation, acrylation, glycosylation, methylation or farnesylation.

The sLRP6E1E2 polypeptide or its variants can be prepared by preparationfrom its natural source, chemical synthesis (Merrifleld, J. Amer chem.Soc. 85:2149-2156, 1963) or recombinant methods based on DNA sequences(Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)).

In still another aspect of this invention, there is provided acomposition for preventing or treating cancer, comprising a nucleotidesequence encoding the amino acid sequence of SEQ ID NO:2.

In still another aspect of this invention, there is provided a methodfor preventing or treating cancer comprising administering to a subjectin need thereof a pharmaceutically effective amount of a nucleotidesequence encoding the amino acid sequence of SEQ ID NO:2.

According to a concrete embodiment, the nucleotide sequence comprisesthe nucleotide sequence of SEQ ID NO:1.

The SEQ ID NO:1 is the nucleotide sequence of sLRP6E1E2 which iscomposed of the E1 and E2 extracellular domains (Wnt-binding sites) ofLRP6

It would be obvious to the skilled artisan that the nucleotide sequencesused in this invention are not limited to those listed in the appendedSequence Listings.

For nucleotide sequences, the variations may be purely genetic, i.e.,ones that do not result in changes in the protein product. This includesnucleic acids that contain functionally equivalent codons, or codonsthat encode the same amino acid, such as six codons for arginine orserine, or codons that encode biologically equivalent amino acids.

Considering biologically equivalent variations described hereinabove,the nucleic acid molecule of this invention may encompass sequenceshaving substantial identity to them. Sequences having the substantialidentity show at least 80%, more preferably at least 90%, mostpreferably at least 95% similarity to the nucleic acid molecule of thisinvention, as measured using one of the sequence comparison algorithms.Methods of alignment of sequences for comparison are well-known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J.Mol. Bio. 48:443(1970); Pearson and Lipman, Methods in Mol. Biol. 24:307-31(1988); Higgins and Sharp, Gene 73:237-44(1988); Higgins andSharp, CABIOS 5:151-3(1989) Corpet et al., Nuc. Acids Res.16:10881-90(1988) Huang et al., Comp. Appl. BioSci. 8:155-65(1992) andPearson et al., Meth. Mol. Biol. 24:307-31(1994). The NCBI Basic LocalAlignment Search Tool (BLAST) [Altschul et al., J. Mol. Biol.215:403-10(1990)] is available from several sources, including theNational Center for Biological Information (NBCI, Bethesda, Md.) and onthe Internet, for use in connection with the sequence analysis programsblastp, blasm, blastx, tblastn and tblastx. It can be accessed athttp://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determinesequence identity using this program is available athttp://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to a concrete embodiment, the nucleotide sequence is containedin a gene delivery system.

The term “gene delivery system” as used herein, refers to any forms ofcarriers that harbor and transport exogenous nucleic acid molecules to ahost cell or tissue. The ideal gene delivery system should be harmlessto human body, suitable for mass production, and capable of effectivetransportation of the target gene.

According to a more concrete embodiment, the gene delivery system isplasmid, recombinant adenovirus, adeno-associated virus (AAV),retrovirus, lentivirus, herpes simplex virus, vaccinia virus, measlesvirus, poxvirus, Semliki Forest virus, polymer, nanomaterial, lipospmeor niosome.

The nucleotide sequence of this invention may be applied to all genedelivery system commonly used for gene therapy, concretely, plasmid,adenovirus (Lockett L J, et al., Clin. Cancer Res., 3:2075-2080(1997)),adeno-associated virus (AAV, Lashford L S., et al., Gene TherapyTechnologies, Applications and Regulations Ed. A. Meager, 1999),retrovirus (Gunzburg W H, et al., Retroviral vectors. Gene TherapyTechnologies, Applications and Regulations Ed. A. Meager, 1999),lentivirus (Wang G. et al., J. Clin. Invest. 104(11):R55-62(1999)),herpes simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA,92:1411-1415(1995)), vaccinia virus (Puhlmann M. et al., Human GeneTherapy, 10:649-657(1999)), poxvirus (GCE, NJL, Krupa M, Esteban M., Thepoxvirus vectors MVA and NYVAC as gene delivery systems for vaccinationagainst infectious diseases and cancer Curr Gene Ther8(2):97-120(2008)), reovirus, measles virus, Semliki Forest virus,polymer (Hwang et al., In vitro and In vivo Transfection Efficiency ofHuman Osteoprotegerin Gene using Non-Viral Polymer Carriers., Korean J.Bone Metab. 13(2):119-128(2006)), lipospme (Methods in MolecularBiology, Vol 199, S. C. Basu and M. Basu (Eds.), Human Press 2002),nanomaterial or noisome. Most concretely, the gene delivery system ofthis invention is constructed by incorporating the nucleotide sequenceof sLRP6E1E2 to adenoviruses.

i. Adenovirus

Adenovirus has been usually employed as a gene delivery vector becauseof its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. Both ends of the viral genomecontains 100-200 bp ITRs (inverted terminal repeats), which are ciselements necessary for viral DNA replication and packaging. The E1region (E1A and E1B) of genome encodes proteins responsible for theregulation of transcription of the viral genome and a few cellulargenes. The E2 region (E2A and E2B) encodes proteins responsible forviral DNA replication.

Of adenoviral vectors developed so far, the replication incompetentadenovirus having the deleted E1 region is usually used. The deleted E3region in adenoviral vectors may provide an insertion site fortransgenes (Thimmappaya, B. et al., Cell, 31:543-551(1982); and Riordan,J. R. et al., Science, 245:1066-1073(1989)).

Therefore, the sequence of this invention may be inserted into the DApromoter region. The term “deletion” with reference to viral genomesequences encompasses whole deletion and partial deletion as well.

According to a concrete embodiment, the recombinant adenovirus comprisesdeleted E1B and E3 region and the nucleotide sequence of SEQ ID NO:1 isinserted into the deleted E1B and E3 region.

In nature, adenovirus can package approximately 105% of the wild-typegenome, providing capacity for about 2 extra kb of DNA (Ghosh-Choudhuryet al., EMBO J., 6:1733-1739(1987)). In this regard, the foreignsequences described above inserted into adenovirus may be furtherinserted into adenoviral wild-type genome.

The foreign genes delivered by the present adenoviral gene deliverysystem are episomal, and therefore, have low genotoxicity to host cells.Therefore, gene therapy using the adenoviral gene delivery system ofthis invention may be considerably safe.

ii. Retrovirus

Retroviruses capable of carrying relatively large exogenous genes havebeen used as viral gene delivery vectors in the senses that theyintegrate their genome into a host genome and have broad host spectrum.

In order to construct a retroviral vector, the cytokine gene is insertedinto the viral genome in the place of certain viral sequences to producea replication-defective virus. To produce virions, a packaging cell linecontaining the gag, pol and env genes but without the LTR (long terminalrepeat) and ψ components is constructed (Mann et al., Cell,33:153-159(1983)). When a recombinant plasmid containing the cytokinegene, LTR and ψ is introduced into this cell line, the ψ sequence allowsthe RNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubinstein “Retroviral vectors,” In: Vectors: A survey of molecularcloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham:Butterworth, 494-513(1988)). The media containing the recombinantretroviruses is then collected, optionally concentrated and used forgene delivery system.

A successful gene transfer using the second-generation retroviral vectorhas been reported. Kasahara et al. (Science, 266:1373-1376(1994))prepared variants of moloney murine leukemia virus in which the EPO(erythropoietin) sequence is inserted in the place of the enveloperegion, consequently, producing chimeric proteins having novel bindingproperties. Likely, the present gene delivery system can be constructedin accordance with the construction strategies for the second-generationretroviral vector.

iii. AAV Vector

Adeno-associated viruses are capable of infecting non-dividing cells andvarious types of cells, making them useful in constructing the genedelivery system of this invention. The detailed descriptions for use andpreparation of AAV vector are found in U.S. Pat. Nos. 5,139,941 and4,797,368. Research results for AAV as gene delivery systems aredisclosed in LaFace et al, Viology, 162:483486 (1988), Zhou et al., Exp.Hematol. (NY), 21:928-933 (1993), Walsh et al, J. Clin. Invest.,94:1440-1448(1994) and Flotte et al., Gene Therapy, 2:29-37(1995).Recently, an AAV vector has been approved for Phase I human trials forthe treatment of cystic fibrosis.

Typically, a recombinant AAV virus is made by cotransfecting a plasmidcontaining the gene of interest (i.e., cytokine gene) flanked by the twoAAV terminal repeats (McLaughlin et al., J. Virol., 62:1963-1973(1988);Samulski et al., J. Virol., 63:3822-3828(1989)) and an expressionplasmid containing the wild type AAV coding sequences without theterminal repeats (McCarty et al., J. Virol., 65:2936-2945(1991)).

iv. Other Viral Vectors

Other viral vectors may be employed as a gene delivery system in thepresent invention. Vectors derived from viruses such as vaccinia virus(Puhlmann M. et al., Human Gene Therapy, 10:649-657(1999); Ridgeway,“Mammalian expression vectors,” In: Vectors: A survey of molecularcloning vectors and their uses. Rodriguez and Denhardt, eds. Stoneham:Butterworth, 467-492(1988); Baichwal and Sugden, “Vectors for genetransfer derived from animal DNA viruses: Transient and stableexpression of transferred genes,” In: Kucherlapati R, ed. Gene transfer.New York: Plenum Press, 117-148(1986) and Coupar et al., Gene,68:1-10(1988)), lentivirus (Wang G. et al., J. Clin. Invest.,104(11):R55-62(1999)), herpes simplex virus (Chamber R., et al., Proc.Natl. Acad. Sci USA, 92:1411-1415(1995)), poxvirus (GCE, NJL, Krupa M,Esteban M., The poxvirus vectors MVA and NYVAC as gene delivery systemsfor vaccination against infectious diseases and cancer Curr Gene Ther8(2):97-120(2008)), reovirus, measles virus, Semliki Forest virus, maybe used in the present delivery systems for transferring the gene ofinterest into cells.

v. Polymer

Polymer vectors widely used as non-viral gene delivery system includePEG, PEI, PLL, gelatin, chitosan (Carreno G B, Duncan R. Evaluation ofthe biological properties of soluble chitosan and chitosan microspheres.Int J Pharm 148:231-240(1997)) PLL(poly-LLysine)(Maruyama A, Ishihara T,Kim J S, Kim S W, Akaike T. Nanoparticle DNA carrier with poly(L-lysine) grafted polysaccharide copolymer and poly (D,Llactide).Bioconjugate Chem 8:735-742(1997)) and PEI (polyethyleneamine) (AbdallahB, Hassan A, Benoist C, Goula D, Behr J P, Demeneix B A. A powerfulnonviral vector for in vivo gene transfer into the adult mammalianbrain: Polyethyleneimine. Human Gene Ther 7:1947-1954(1996)). Theadvantages of polymer vector lie on low occurrence of immune responseand acute toxicity, and simple preparation enabling mass production.

vi. Lipospme and Niosome

Liposomes are formed spontaneously when phospholipids are suspended inan excess of aqueous medium. Liposome-mediated nucleic acid delivery hasbeen very successful as described in Nicolau and Sene, Biochim. Biophys.Acta, 721:185-190(1982) and Nicolau et al., Methods Enzymol.,149:157-176(1987). Example of commercially accessible reagents fortransfecting animal cells using liposomes includes Lipofectamine (GibcoBRL). Liposomes entrapping the nucleotide sequence of interest interactwith cells by mechanism such as endocytosis, adsorption and fusion andthen transfer the sequences into cells.

Niosome is a bilayer vehicle composed of mixture of non-ionic surfactantand cholesterol which may encapsulate polar and non-polar material.Niosome has osmotic activity, higher stability compared to liposome. Thesurfactant used for the preparation of liposome is more easy in storageand handling.

The introduction into host cell of the gene delivery system can beperformed through various methods known to those skilled in the art.

Where the present gene delivery system is constructed on the basis ofviral vector construction, the contacting is performed as conventionalinfection methods known in the art. The infection of hosts using viralvectors is well described in the above-cited publications.

Where the gene delivery system of the present invention is nakedrecombinant DNA molecule or plasmid, it can be injected into the hostcell by micro-injection (Capecchi, M. R., Cell, 22:479(1980); andHarland and Weintraub, J. Cell Biol. 101:1094-1099(1985)), calciumphosphate precipitation method (Graham, F. L. et al., Virology,52:456(1973); and Chen and Okayama, Mol. Cell. Biol. 7:2745-2752(1987)),electroporation (Neumann, E. et al., EMBO J., 1:841(1982); and Tur-Kaspaet al., Mol. Cell Biol., 6:716-718(1986)), liposome-mediated infectionmethod (Wong, T. K. et al., Gene, 10:87(1980); Nicolau and Sene,Biochim. Biophys. Acta, 721:185-190 (1982); and Nicolau et al., MethodsEnzymol., 149:157-176 (1987)), DEAE-dextran treatment (Gopal, Mol. CellBiol., 5:1188-1190 (1985)), and gene bombardment (Yang et al., Proc.Natl. Acad. Sci., 87:9568-9572 (1990)).

According to the most concrete embodiment, the gene delivery system isrecombinant adenovirus.

According to a concrete embodiment, the recombinant adenovirus comprisesdeleted E1B and E3 region and the nucleotide sequence of SEQ ID NO:1 isinserted into the deleted E1B and E3 region.

According to a concrete embodiment, the cancer prevented or treated bythe composition of the present invention is lung cancer.

According to a concrete embodiment, the composition of the presentinvention inhibits activation of Wnt signaling pathway in a cancer cell.

According to a more concrete embodiment, the composition of the presentinvention inhibits activation of Wnt signaling pathway in a cancer cellthrough binding of the polypeptide having the amino acid sequence of SEQID NO:2 to Wnt 3a protein.

According to the present invention, sLRP6E1E2-transfected cell showslower level of Wnt 3a and LPR6 proteins compared to controls, suggestingthat LPR6 E1-E2 domains of sLRP6E1E2 effectively bind to Wnt 3a, therebyinhibit Wnt signaling which is involved in cancer progression.

Since the composition of the present invention is highly effective inreducing cancer cell growth and proliferation, and inducing apoptosis ofcancer cells, it is useful in treating tumor-related diseases, includingstomach cancer, lung cancer, breast cancer, ovarian cancer, livercancer, brain cancer, prostate cancer, sarcoma, bronchogenic cancer,nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladdercancer, colon cancer, and uterine cervical cancer.

Concretely, the cancer prevented or treated by the composition of thepresent invention is lung cancer.

The term “treatment” as used herein, refers to (i) prevention oftumorigenesis; (ii) suppression and curing of tumor-related diseases ordisorders by eradicating tumor cells; and (iii) alleviation oftumor-related diseases or disorders by eradicating tumor cells.

The pharmaceutically acceptable carrier which may be contained in thecomposition of the present invention is commonly used in pharmaceuticalformulations including but is not limited to, lactose, dextrose,sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate,arginate, gelatin, potassium silicate, microcrystalline cellulose, DMSO,polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose,methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesiumstearate, and mineral oils. The composition of the present invention mayfurther include a lubricant, a humectant, a sweetener, a flavoringagent, an emulsifier, a suspending agent, and a preservative.

The composition of the present invention may be administered orally orparenterally, and preferably, administered parenterally. For parenteraladministration, it may be administered intravenously, intraperitoneally,intramuscularly, intradermally or topically. The composition of thepresent invention may be administered intraperitoneally in ovariancancer, intravenously in liver cancer, injected directly to tumor massin breast cancer, directly administered by rectal injection in coloncancer, and directly administered via catheter in bladder cancer.

A suitable dosage amount of the pharmaceutical composition of thepresent invention may vary depending on pharmaceutical formulationmethods, administration methods, the patient's age, body weight, sex,pathogenic state, diet, administration time, administration route, anexcretion rate and sensitivity for a used pharmaceutical composition andphysicians of ordinary skill in the art can determine an effectiveamount of the pharmaceutical composition for desired treatment.Generally, the pharmaceutical composition of the present inventioncomprises 1×10⁵-1×10¹⁵ pfu/ml of a recombinant adenovirus and 1×10¹⁰ pfuof a recombinant adenovirus is typically injected once every other dayover two weeks.

According to the conventional techniques known to those skilled in theart, the composition of the present invention may be formulated withpharmaceutically acceptable carrier and/or vehicle as described above,finally providing several forms a unit dose form and a multi-dose form.Non-limiting examples of the formulations include, but not limited to, asolution, a suspension or an emulsion in oil or aqueous medium, anextract, an elixir, a powder, a granule, a tablet and a capsule, and mayfurther comprise a dispersion agent or a stabilizer.

The pharmaceutical composition comprising the recombinant adenovirusaccording to the present invention may be utilized alone or incombination with typical chemotherapy or radiotherapy. Such combinationtherapy may be more effective in treating cancer. The chemotherapeuticagents useful for the combination therapy include cisplatin,carboplatin, procarbazine, mechlorethamine, cyclophosphamide,ifosfamide, melphalan, chlorambucil, bisulfan, nikosourea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide,tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastinand methotrexate. Examples of the radiotherapy useful for thecombination therapy include X-ray illumination and γ-ray illumination.

Cell therapy that may be combined with the present invention includesdendritic cells, NK (Natural Killer) cells, TIL (Tumor InfiltratingLymphocytes) and CTL (Cytotoxic T Lymphocytes).

The features and advantages of the present invention will be summarizedas follows:

(a) The present invention provides a composition for preventing ortreating cancer comprising Wnt decoy receptor.

(b) The composition of the present invention or the expression productthereof inhibits cancer generation, growth, proliferation andmetastasis, and induces apoptosis of cancer cells, by binding to Wntligand and blocking ligand-receptor interactions, therefore may beeffectively used as an anti-cancer agent.

The present invention will now be described in further detail byexamples. It would be obvious to those skilled in the art that theseexamples are intended to be more concretely illustrative and the scopeof the present invention as set forth in the appended claims is notlimited to or by the examples.

EXAMPLE

Materials and Methods

Materials

Polyclonal antibodies against MAPK kinase (MEK1/2), p44/42mitogen-activated protein kinase (MAPK; Erk1/2), mTOR,phosphatidylinositol 3-kinase (PI3K) and Akt, and monoclonal antibodiesagainst Wnt3a, Dvl2, Axin, glycogen synthase kinase (GSK3-β), poly(ADP-ribose) polymerase (PARP), and cleaved caspase-3 were purchasedfrom Cell Signaling Technology (Beverly, Mass.). Antibodies againstepithelial-to-mesenchymal transition (EMT)-related molecules β-cateninand E-cadherin were obtained from Cell Signaling Technology. Antibodiesagainst cyclin D1 (H-295), cytochrome c (C-20 for Western blotanalysis), and LRP6 (C-10), and protein A/G agarose beads were purchasedfrom Santa Cruz Biotechnology (Santa Cruz, Calif.). Monoclonal antibodyagainst caspase-3 was from StressGen Biotechnologies (Victoria, BC).Polyclonal antibody against cytochrome c (6H2.B4 forImmunohistochemistry) was from BD Pharmingen (San Diego, Calif.). AlexaFluor 488-conjugated and Alexa Fluor 568-conjugated anti-rabbit IgGantibodies were obtained from Invitrogen (Carlsbad, Calif.). DAPI (1μg/ml), Hoechst 33342, and tetramethylrhodamine isothiocyanate(TRITC)-conjugated phalloidin were from Sigma (St. Louis, Mo.). PurifiedWnt3a protein was purchased from R&D Systems (Minneapolis, Minn.).

Generation of Adenoviral Vectors Expressing Soluble LRP6 Receptor

To study the biochemical function of soluble LRP6 receptor (sLRP6E1E2),we generated constructs of the E1 and E2 extracellular domains(Wnt-binding sites) of LRP6 (17) and FLAG-tagged sLRP6E1E2 was subclonedinto a pCA14 shuttle vector (18). This pCA14-sLRP6E1E2 vector wasco-transformed with a replication-incompetent adenovirus 5/35 chimericvector (dE1-k35) or replication-competent chimeric oncolytic adenovirusvector (RdB-k35) (19), generating pdE1-k35/sLRP6E1E2 andpRdB-k35/sLRP6E1E2, respectively. These recombinant plasmids weretransfected into HEK293 cells to generate dE1-k35/sLRP6E1E2 andRdB-k35/sLRP6E1E2. The replication-incompetent dE1-k35/LacZ andreplication-competent oncolytic RdB-k35 vectors were used as negativecontrols (20) (FIG. 1A). All viruses were obtained as previouslydescribed (21).

Luciferase Reporter Assay for β-Catenin Activity

TOPflash and FOPflash luciferase reporter vectors (UpstateBiotechnology, Lake Placid, N.Y.) were used to measure β-catenin/T-cellfactor (TCF) signaling activity. A549, H322, and H460 cells were seededand transfected with 0.3 μg TOPflash (containing wild-type TCF bindingsites) or FOPflash (containing mutated TCF binding sites) negativecontrol with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (20, 50 MOI). After 12hr, the medium was replaced with 1% DMEM with or without 100 ng/ml ofWnt3a, and the cells were incubated for another 24 hr. Cell extract wasthen analyzed using the Dual-Luciferase Reporter Assay System (Promega,Madison, Wis.).

Cell Proliferation Assay

The cell proliferation assay was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay(Sigma) (19). A549 and H322 cells were seeded in 24-well plates (2×10⁴cells/well). After 24 hr, cells were treated with PBS, dE1-k35/LacZ, ordE1-k35/sLRP6E1E2. The next day, cells were stimulated with or withoutrecombinant Wnt3a (100 ng/ml) for an additional 48 hr. Absorbance at 540nm was read on a microplate reader.

Western Blotting and Immunoprecipitation

Cells cultured in DMEM with 1% fetal bovine serum in 100-mm plates weretransduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2. The next day, cellswere treated with or without Wnt3a (100 ng/ml) for 16 hr. Immunoblottingwas performed as described previously (19).

Immunofluorescence Assay

For immunofluorescence microscopy, cultured cells were fixed andpermeabilized. The samples were blocked and then incubated withE-cadherin, β-catenin, or anti-cytochrome c primary antibodies. Stainingwas visualized by Alexa Flour 488-conjugated goat anti-rabbit IgGsecondary antibody. The final antibody treatment also containedTRITC-conjugated phalloidin and Hoechst 33342 or DAPI stain (both at 1μg/ml, Sigma) for nuclear staining. The cells were viewed under aconfocal laser-scanning microscope (LSM510, Carl Zeiss Microlmaging,Thornwood, N.Y.).

Mitochondrial Fractionation and Western Blotting

Mitochondrial fractions were prepared using the Qproteome mitochondriaisolation kit (QIAGEN, Hilden, Germany) following the manufacturer'sinstructions. Cells washed and suspended with ice-cold lysis buffer.After 10-min incubation, lysate was centrifuged, and the supernatantcontaining cytosolic proteins was removed. The pellet containing nuclei,cell debris, and unbroken cells was resuspended with ice-cold disruptionbuffer and centrifuged, and the supernatant (microsomal fraction) wastransferred to a clean microtube. The resulting pellet containingmitochondria was washed with the mitochondria storage buffer andcentrifuged. Western blotting was performed with the rabbitanti-cytochrome c antibody using the procedure described above.

Cytochrome c Immunostaining

A549 cells were plated in two-chamber slides (3×10⁴ cells/chamber; Nunc,Naperville, Ill.) and transduced with PBS, dE1-k35/LacZ, ordE1-k35/sLRP6E1E2 after treatment with or without Wnt3a. The next day,cells were fixed and incubated with medium containing 250 nM MitoTrackerRed mitochondria stain (Molecular Probes, Eugene, Oreg.) for 30 min atroom temperature. Cells were then incubated with 0.5 mg/mlanti-cytochrome c antibody. Next day, staining was visualized by AlexaFlour 488-conjugated goat anti-mouse IgG antibody. After washing, theslides were stained with Hoechst 33258 (1 mg/ml) for nuclear staining.

Anti-Tumor Effects in Human Xenograft Model

Human non-small cell lung cancer xenograft was established in 6- to8-week-old male athymic nu/nu mice (Charles River Japan, Yokohama,Japan) by subcutaneous implantation of 1×10⁷ H460 cells in the abdomen.When tumor volumes reached approximately 80-100 mm³, the mice weredivided five groups with similar mean tumor volumes. Adenoviral vectorswere administered intratumorally (2×10¹⁰ viral particles/mouse) on thefirst day of treatment (day 1) and days 3 and 5. Tumor volume (V) wascalculated as V=0.52×a²×b (a, smallest superficial diameter; b, largestsuperficial diameter).

Tumor Histology and Immunohistochemistry

Tumor tissue was fixed and embedded in paraffin wax for histologicexamination and immunohistochemical staining. Representative sectionswere stained with hematoxylin and eosin and examined by lightmicroscopy. To quantify capillary density and Wnt expression, the tumorsections were stained with anti-mouse CD31 IgG (BD Pharmingen),anti-rabbit β-catenin IgG (Cell Signaling Technology), or anti-mouseWnt3a IgG (Santa Cruz Biotechnology). Positive immunoreactivity wasvisualized with ABC-peroxidase kits (ChemMate™ DAKO Envision™ Detectionkit; DAKO).

Migration and Invasion Assay

In vitro migration assays were performed as described previously (23).Conditioned media was obtained from A549 cells transduced with PBS,dE1-k35/LacZ, or dE1-k35/sLRP6E1E2 after treatment with or without Wnt3aand placed in the bottom Transwell chamber. A549 cells were then platedon the upper chamber (7×10⁴ cells/well). Cultures were incubated at 37°C. for 4 hr, fixed, and stained with hematoxylin and eosin. In vitroMatrigel invasion assays were performed using bio-coat cell migrationchambers. Filters (8-μm pore) were coated with Matrigel basementmembrane matrix (37 mg/filter; BD Biosciences, San Jose, Calif.), andthe experiment was performed as described for the cell migration assay.

Reverse Transcription (RT)-PCR

Briefly, total RNA (1 μg) was isolated using the RNeasy Mini Kit(QIAGEN, Valencia, Calif.) and cDNA was synthesized with oligo-dTprimers and M-MLV reverse transcriptase (Invitrogen). Polymerase chainreaction (PCR) was carried out using cDNA (25 ng) as a template and thefollowing PCR primers: matrix metalloproteinase (MMP)2, forward5′-CTCAGATCCGTGGTGAGATCT-3′, reverse 5′-CTTTGGTTCTCCAGCTTCAGG-3′, MMP9,forward 5′-ATCCAGTTTGGTGTCGCGGAGC-3′, reverse5′-GAAGGGGAAGACGCACAGCT-3′, E-cadherin, 5′-ACGATGATGTGAACACCTACA-3′,reverse 5′-ATGCCATCGT TGTTCACTGCA-3′, Vimentin, forward5′-TGGCACGTCTTGACCTTGAA-3′, reverse 5′-GGTCATCGTGATGCTGAGAA-3′,β-catenin, forward 5′-GCTGATTTGATGGAGTTGGA-3′, reverse5′-TCAGCTACTTGTTCTTGAGTGAA-3′, β-actin, forward5′-CCTTCCTGGGCATGGAGTCCT-3′, reverse 5′-GGAGCAATGATCTTGATCTT-3′.

Statistical Analysis

Results are expressed as mean±standard error of the mean (SEM). Groupresults were compared by one-way analysis of variance, followed by posthoc Student's t-test for unpaired observations or Bonferroni'scorrection for multiple comparisons when appropriate. P<0.05 wasconsidered significant.

Results

Soluble Wnt Decoy Receptor is Expressed in Lung Cancer Cell Lines andBinds to Wnt3a

Endogenous Wnt3a levels were assessed in six non-small cell lung cancercell lines (A549, H322, H596, H460, H358, and H2009) by western blotanalysis. Wnt3a was more strongly expressed in H322, H460, and H2009cells than in other cell lines (FIG. 1B); therefore, H322 and H460 cellswere selected to evaluate the ability of the soluble Wnt decoy receptor(sLRP6E1E2) to inhibit Wnt signaling. Expression of sLRP6E1E2 fromdE1-k35/sLRP6E1E2-transduced A549 cells was confirmed by western blotanalysis using anti-FLAG and anti-LRP6 antibodies (FIG. 1C). Secretionof sLRP6E1E2 from dE-k35/sLRP6E1E2-transduced cells was dose-dependent.

Next, binding of sLRP6E1E2 to Wnt3a was assessed in cells transducedwith dE1-k35/LacZ or dE1-k35/sLRP6E1E2. Cell lysates wereimmunoprecipitated with anti-Wnt3a or anti-LRP6 antibodies and analyzedby western blot. As shown in FIG. 1D, Wnt3a and LRP6 protein levels werelower in cells transduced with dE1-k35/sLRP6E1E2 than in cellstransduced with dE1-k35/LacZ, indicating that the LRP6 E1-E2 domainsefficiently bind to Wnt3a.

Decoy Wnt Receptor Decreases Cytosolic β-Catenin Level and TCFTranscriptional Activity

Because sLRP6E1E2 binds to Wnt3a, we determined its effect on β-cateninusing a luciferase reporter system activated by β-catenin/TCF (24). Asshown in FIG. 2A, luciferase activity was low in A549 cells transducedwith dE1-k35/LacZ or dE1-k35/sLRP6E1E2 in the absence of Wnt3a. Wnt3atreatment increased luciferase expression approximately 7- to 8-fold incontrol cells, but not in dE1-k35/sLRP6E1E2-transduced cells. In theabsence of Wnt3a, luciferase activity was reduced by dE1-k35/sLRP6E1E2in H460 (48%) and H322 (12%) cells compared with dE1-k35/LacZ controls(FIG. 2B; P<0.05). Wnt3a stimulation increased luciferase activity inH460 (53%) and H322 (102%) cells transduced with dE1-k35/LacZ, butluciferase activity was significantly lower indE1-k35/sLRP6E1E2-transduced H460 (48%) and H322 (52%) cells comparedwith dE1-k35/LacZ (P<0.05).

To evaluate the effect of sLRP6E1E2 on β-catenin localization, immunefluorescence staining was performed in H322 cells treated with PBS ortransduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2. In the absence ofWnt3a, β-catenin staining was restricted primarily to cell-cell contactsites in all groups. Upon Wnt3a stimulation, control cells (PBS anddE1-k35/LacZ) showed reduced β-catenin localization at the plasmamembrane, especially at cell-cell junctions, and increased β-cateninlevels in the cytosol and nucleus. In contrast,dE1-k35/sLRP6E1E2-transduced cells showed lower levels of cytosolicβ-catenin, and higher levels of membrane-associated β-catenin (FIG. 2C).Results of these functional studies demonstrate that interactionsbetween sLRP6E1E2 and Wnt may be sufficient to block Wnt signaling.

Decoy Wnt Receptor sLRP6E1E2 Inhibits Lung Cancer Cell Proliferation

The Wnt pathway regulates a wide range of cellular functions includingproliferation (25). To test the effects of sLRP6E1E2 on proliferation ofA549 and H322 cells in vitro, cells were treated with PBS or transducedwith dE1-k35/LacZ or dE1-k35/sLRP6E1E2. At 72 hr after transduction withdE1-k35/sLRP6E1E2 (20 MOI), cell proliferation was reduced by 39% inA549 cells and 51% in H322 cells compared with dE1-k35/LacZ-transducedcontrols. Wnt3a stimulation increased proliferation approximately 10-20%in control cells, but had no apparent effect ondE1-k35/sLRP6E1E2-transduced cells. Proliferation was 54% lower in A549cells and 61 lower in H322 dE1-k35/sLRP6E1E2-transduced cells thandE1-k35/LacZ-transduced cells (P<0.001; FIG. 3A).

To characterize signaling pathways involved in the anti-proliferativeaction of sLRP6E1E2, we examined its effects on canonical Wnt signaling.As shown in FIG. 3B, Dvl2 and Axin protein levels in control cells (PBSand dE1-k35/LacZ) were increased by Wnt3a, but were apparently unalteredby Wnt3a in dE1-k35/sLRP6E1E2-transduced cells. Similarly, cyclin D1expression was slightly increased in control cells following Wnt3astimulation, but slightly decreased in dE1-k35/sLRP6E1E2-transducedcells. GSK3β levels also appeared slightly decreased after Wnt3atreatment.

Wnt plays a fundamental role in proliferation by activating ERK andPI3K-Akt pathways (26). We therefore investigated whether sLRP6E1E2 candownregulate these pathways in H460 cells. We found that basal levels ofMEK, ERK1/2, and survivin were lower in dE1-k35/sLRP6E1E2-transducedcells than in control cells (FIG. 3C). These proteins were upregulatedby Wnt3a treatment, but levels were still considerably lower indE1-k35/sLRP6E1E2-transduced cells than in the controls. Expression ofmTOR, PI3K, and Akt was not affected by Wnt3a stimulation, and was lowerin dE1-k35/sLRP6E1E2-transduced cells than controls. Taken together,these results suggest that sLRP6E1E2 exerts antiproliferative actions byinhibiting Wnt signaling via MEK-ERK and PI3K-Akt pathways.

Decoy Wnt Receptor sLRP6E1E2 Induces Apoptosis

Wnt signaling can prevent apoptosis and promote cellular proliferationand survival (27). To characterize the molecular mechanisms by whichsLRP6E1E2 inhibits non-small cell lung cancer proliferation, weevaluated the effects of sLRP6E1E2 on apoptosis. At 3 days afterdE1-k35/sLRP6E1E2 transduction, we observed that A549, H1299, and H358cells gradually detached from the culture dish and became rounder andsmaller than attached cells (FIG. 4A), suggesting that sLRP6E1E2 inducedapoptosis. Evidence of apoptosis was sought by looking for nuclearapoptotic bodies (data not shown), and then assessed using the TUNELassay to detect internucleosomal DNA fragmentation (28). As shown inFIG. 4B, more TUNEL-positive cells were observed amongdE1-k35/sLRP6E1E2-transduced cells than among control cells in thepresence or absence of Wnt3a. Quantitation of TUNEL staining revealedthat the rate of apoptosis was approximately 1.9-fold higher (withoutWnt3a) and 2.8-fold higher (with Wnt3a) in dE1-k35/sLRP6E1E2-transducedcells than in dE1-k35/LacZ-transduced controls (P<0.001) (FIG. 4C).

We next evaluated regulators of apoptosis, of which the caspase familyand cytochrome c are the best characterized. In the absence and presenceof Wnt3a, full-length 116-kDa PARP protein was reduced and 85-kDacleavage fragments were increased in dE1-k35/sLRP6E1E2-transduced cells(FIG. 4D). Levels of the cleaved (active) form of caspase-3 were alsomarkedly increased by sLRP6E1E2. As shown in FIG. 4E,dE1-k35/sLRP6E1E2-transduced cells also showed increased cytosoliccytochrome c and decreased microsomal cytochrome c. Stimulation withWnt3a produced similar effects. To further investigate cytochrome clocalization, immunofluorescence was performed. PBS-treated anddE1-k35/LacZ-transduced cells displayed punctuate cytoplasmic stainingof cytochrome c, consistent with mitochondrial localization. Incontrast, cells expressing sLRP6E1E2 exhibited mostly diffusecytoplasmic cytochrome c staining, consistent with translocation frommitochondria to cytoplasm (FIG. 4F).

Decoy Wnt Receptor sLRP6E1E2 Inhibits Tumor Xenograft Growth

We next evaluated the ability of sLRP6E1E2 to inhibit tumor growth in amouse xenograft model. Tumors were generated by subcutaneous injectionof H460 cells into the abdominal region of nude mice. When tumorsreached a mean size of 80-100 mm³, they were injected with PBS, dE1-k35,RdB-k35, dE1-k35/sLRP6E1E2, or RdB-k35/sLRP6E1E2 on days 1, 3, and 5.FIG. 5A shows that the volume of tumors injected withsLRP6E1E2-expressing vectors was significantly lower than that ofcorresponding controls. After 25 days, tumors treated with PBS reached amean volume of 3883.1±418.08 mm³, and tumors treated with dE1-k35 andRdB-k35 reached 3388.1±226.9 mm³ and 1991±311.8 mm³, respectively. Incontrast, tumor growth was strongly suppressed in mice injected withdE1-k35/sLRP6E1E2 (1645.3±353.6 mm³; P<0.05 compared with PBS or dE1-k35groups) or RdB-k35/sLRP6E1E2 (923.3±180.4 mm³; P<0.01 compared with PBSor RdB-k35 groups).

To evaluate the biological effects of sLRP6E1E2 in tumor tissue, tumorswere harvested 3 days after the final adenovirus injection. Analysis ofadenoviral E1A protein expression revealed that RdB-k35 andRdB-k35/sLRP6E1E2 had replicated and spread through the (FIG. 5B, E1A).Immunohistochemical analysis of sLRP6E1E2 (FIG. 5B, FLAG) showed thatits expression was more widespread in RdB-k35/sLRP6E1E2-treated tumorsthan in dE1-k35/sLRP6E1E2-treated tumors, indicating that the oncolyticadenovirus more efficiently expressed sLRP6E1E2 than thereplication-incompetent adenovirus, contributing to its superiorantitumor actions.

Anti-Proliferative and Apoptotic Effects of sLRP6E1E2-Expressing Vectorsin H460 Xenografts

To assess the effects of sLRP6E1E2 on tumor xenograft growth in mice,tumor samples were analyzed by Ki-67 immunostaining for proliferatingcells and TUNEL staining for apoptotic cells. We found that Ki-67expression was reduced and TUNEL-positive cells were increased in tumorstreated with dE1-k35/sLRP6E1E2 or RdB-k35/sLRP6E1E2 compared withcorresponding controls (FIG. 5C). We also detected more TUNEL-positivecells in RdB-k35/sLRP6E1E2-treated tumors than indE1-k35/sLRP6E1E2-treated tumors, consistent with previous results. Todetermine whether the smaller sLRP6E1E2-treated tumors exhibited reducedneovascularization, microvessel density was assessed by CD31 staining.Fewer endothelial cells and vessel structures was observed in tissuesinjected with E1-expressing oncolytic adenoviruses (RdB-k35 andRdB-k35/sLRP6E1E2) than PBS-treated tumors (P<0.05), whereas nosignificant decrease in vascular density was observed in tumors injectedwith dE1-k35 or dE1-k35/sLRP6E1E2 (FIGS. 5D & 5E). Further, vesseldensity in tumors injected with sLRP6E1E2-expressing adenoviruses didnot differ from their corresponding controls, suggesting that theantitumor properties of sLRP6E1E2 were not mediated by anti-angiogeniceffects.

To further investigate the role of Wnt signaling in the antitumoractions of sLRP6E1E2-expressing adenoviruses, Wnt and β-cateninlocalization in tumor tissue was evaluated. High endogenous expressionof β-catenin and Wnt was observed in tumor tissues treated with PBS orcontrol vectors (dE1-k35 and RdB-k35) (FIG. 5F), but was significantlyreduced by sLRP6E1E2-expressing vectors, suggesting that blockade of Wntsignaling in tumor cells was an important contributor to slower tumorgrowth.

Wnt Treatment Results Altered Cell Morphology and Induces EMT in TumorCells

EMT is an important process in tumor development, and the Wnt/β-cateninsignal pathway may play an important role in this process. Therefore, weinvestigated whether Wnt3a could induce EMT in H322 cells. We found thatcells became elongated and spindle-shaped 1 day after Wnt3a treatment,resembling the morphology of mesenchymal cells (FIG. 6A). We alsoobserved increased expression of mesenchymal markers Vimentin andβ-catenin with a concomitant decrease in epithelial marker E-cadherin(FIG. 6B). Immunofluorescence staining revealed that cytokeratin andE-cadherin levels were dramatically reduced in cell-cell contacts afterWnt3a treatment (FIG. 6C).

sLRP6E1E2 Modulates EMT-Related Marker Expression and MMP-2/MMP-9Activity.

Acquisition of migratory properties by cancer cells is important formetastatic tumor cell spread (29). Because increasing Wnt3a appeared toenhance motility and invasiveness, we asked whether interfering with theWnt signaling pathway by expressing sLRP6E1E2 would inhibit in vitromotility and invasion. We examined the effect of sLRP6E1E2 on A549 cellsusing transwell motility and matrigel invasion assays. We collectedconditioned medium from PBS-treated, dE1-k35/LacZ-transduced, anddE1-k35/sLRP6E1E2-transduced cells after treatment with or withoutWnt3a. Conditioned medium from dE1-k35/sLRP6E1E2-transduced cellsinhibited migration by 12.4% (without Wnt3a) and 23.8% (with Wnt3a)compared with conditioned medium from dE1-k35/LacZ-transduced cells(P<0.001) (FIG. 7A). Similarly, conditioned medium fromdE1-k35/sLRP6E1E2-transduced cells inhibited invasion by 34.2% (withoutWnt3a) and 56.2% (with Wnt3a) compared with conditioned medium fromdE1-k35/LacZ-transduced cells (FIG. 7B). E-cadherin expression and actinfilaments were also decreased by Wnt3a, but were increased indE1-k35/sLRP6E1E2-transduced cells compared with controls with orwithout Wnt3a treatment (FIG. 7C).

Invasion and metastasis of malignantly transformed cells involvedegradation of extracellular matrix by matrix metalloproteinases (MMPs).We therefore examined the effect of sLRP6E1E2 on expression of MMP-2 andMMP-9, which play a critical role in angiogenesis, tumor growth, andmetastasis. As shown in FIG. 7D, Wnt3a stimulation upregulated MMP-2 andMMP-9 in PBS-treated and dE1-k35/LacZ-transduced A549 cells, butdE1-k35/sLRP6E1E2-transduced cells showed low MMP-2 and MMP-9 expressionwith or without Wnt3a treatment. Taken together, these findings suggestthat sLRP6E1E2 affected multiple Wnt-related pathways in human non-smallcell lung cancer cell lines, leading to reduced cellular invasiveness.

REFERENCES

-   1. Burstein H J, Gelber S, Guadagnoli E, Weeks J C. Use of    alternative medicine by women with early-stage breast cancer. N Engl    J Med 340:1733-9 (1999).-   2. Broxterman H J, Georgopapadakou N H. Anticancer therapeutics: a    surge of new developments increasingly target tumor and stroma. Drug    Resist Updat 10:182-93 (2007).-   3. Liu J R, Opipari A W, Tan L, et al. Dysfunctional apoptosome    activation in ovarian cancer: implications for chemoresistance.    Cancer Res 62:924-31 (2002).-   4. Fukui T, Otani S, Hataishi R, et al. Successful rechallenge with    erlotinib in a patient with EGFR-mutant lung adenocarcinoma who    developed gefitinib-related interstitial lung disease. Cancer    Chemother Pharmacol 65:803-6 (2010).-   5. Lynch T J, Bell D W, Sordella R, et al. Activating mutations in    the epidermal growth factor receptor underlying responsiveness of    non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129-39    (2004).-   6. Nishio M, Koshikawa T, Kuroishi T, et al. Prognostic significance    of abnormal p53 accumulation in primary, resected non-small-cell    lung cancers. J Clin Oncol 14:497-502 (1996).-   7. Nishio M, Koshikawa T, Yatabe Y, et al. Prognostic significance    of cyclin D1 and retinoblastoma expression in combination with p53    abnormalities in primary, resected non-small cell lung cancers. Clin    Cancer Res 3:1051-8 (1997).-   8. Mitsudomi T, Kosaka T, Endoh H, et al. Mutations of the epidermal    growth factor receptor gene predict prolonged survival after    gefitinib treatment in patients with non-small-cell lung cancer with    postoperative recurrence. J Clin Oncol 23:2513-20 (2005).-   9. Nusse R, Theunissen H, Wagenaar E, et al. The Wnt-1 (int-1)    oncogene promoter and its mechanism of activation by insertion of    proviral DNA of the mouse mammary tumor virus. Mol Cell Biol    10:4170-9 (1990).-   10. Beachy P A, Karhadkar S S, Berman D M. Tissue repair and stem    cell renewal in carcinogenesis. Nature 432:324-31 (2004).-   11. Holcombe R F, Marsh J L, Waterman M L, Lin F, Milovanovic T,    Truong T. Expression of Wnt ligands and Frizzled receptors in    colonic mucosa and in colon carcinoma. Mol Pathol 55:220-6 (2002).-   12. DeAlmeida V I, Miao L, Ernst J A, Koeppen H, Polakis P,    Rubinfeld B. The soluble wnt receptor Frizzled8CRD-hFc inhibits the    growth of teratocarcinomas in vivo. Cancer Res 67:5371-9 (2007).-   13. Li Y, Lu W, He X, Schwartz A L, Bu G. LRP6 expression promotes    cancer cell proliferation and tumorigenesis by altering beta-catenin    subcellular distribution. Oncogene 23:9129-35 (2004).-   14. Liu C C, Pearson C, Bu G. Cooperative folding and ligand-binding    properties of LRP6 beta-propeller domains. J Biol Chem 284:15299-307    (2009).-   15. He X, Semenov M, Tamai K, Zeng X. LDL receptor-related proteins    5 and 6 in Wnt/beta-catenin signaling: arrows point the way.    Development 131:1663-1677(2004).-   16. Zhang Y, Wang Y, Li X, et al. The LRP5 high-bone-mass G171V    mutation disrupts LRP5 interaction with Mesd. Mol Cell Biol    24:4677-84(2004).-   17. Forrester W C. The Ror receptor tyrosine kinase family. Cell Mol    Life Sci 59:83-96(2002).-   18. Signorello L B, Cohen S S, Bosetti C, et al. Female survivors of    childhood cancer: preterm birth and low birth weight among their    children. J Natl Cancer Inst 98:1453-61(2006).-   19. Yoo J Y, Kim J H, Kwon Y G, et al. VEGF-specific short hairpin    RNA-expressing oncolytic adenovirus elicits potent inhibition of    angiogenesis and tumor growth. Mol Ther 15:295-302(2007).-   20. Yoon A R, Kim J H, Lee Y S, et al. Markedly enhanced cytolysis    by E1B-19kD-deleted oncolytic adenovirus in combination with    cisplatin. Hum Gene Ther 17:379-90(2006).-   21. Kim J, Kim J H, Choi K J, Kim P H, Yun C O. E1A- and E1B-Double    mutant replicating adenovirus elicits enhanced oncolytic and    antitumor effects. Hum Gene Ther 18:773-86(2007).-   22. Yun C O, Kim E, Koo T, Kim H, Lee Y S, Kim J H.    ADP-overexpressing adenovirus elicits enhanced cytopathic effect by    induction of apoptosis. Cancer Gene Ther 12:61-71(2005).-   23. Yoo J Y, Kim J H, Kim J, et al. Short hairpin RNA-expressing    oncolytic adenovirus-mediated inhibition of IL-8: effects on    antiangiogenesis and tumor growth inhibition. Gene Ther    15:635-51(2008).-   24. Korinek V, Barker N, Morin P J, et al. Constitutive    transcriptional activation by a beta-catenin-Tcf complex in APC−/−    colon carcinoma. Science 275:1784-1787(1997).-   25. Yun M S, Kim S E, Jeon S H, Lee J S, Choi K Y Both ERK and    Wnt/beta-catenin pathways are involved in Wnt3a-induced    proliferation. J Cell Sci 118:313-22(2005).-   26. Kim S E, Lee W J, Choi K Y The PI3 kinase-Akt pathway mediates    Wnt3a-induced proliferation. Cell Signal 19:511-8(2007).-   27. Grotewold L, Ruther U. The Wnt antagonist Dickkopf-1 is    regulated by Bmp signaling and c-Jun and modulates programmed cell    death. EMBO J 21:966-75(2002).-   28. Yang L, Mashima T, Sato S, et al. Predominant suppression of    apoptosome by inhibitor of apoptosis protein in non-small cell lung    cancer H460 cells: therapeutic effect of a novel    polyarginine-conjugated Smac peptide. Cancer Res 63:831-7(2003).-   29. Gupta G P, Massague J. Cancer metastasis: building a framework.    Cell 127:679-95(2006).-   30. Uematsu K, He B, You L, Xu Z, McCormick F, Jablons D M.    Activation of the Wnt pathway in non small cell lung cancer:    evidence of dishevelled overexpression. Oncogene 22:7218-21(2003).-   31. He B, You L, Uematsu K, et al. A monoclonal antibody against    Wnt-1 induces apoptosis in human cancer cells. Neoplasia    6:7-14(2004).-   32. You L, He B, Xu Z, et al. An anti-Wnt-2 monoclonal antibody    induces apoptosis in malignant melanoma cells and inhibits tumor    growth. Cancer Res 64:5385-9(2004).-   33. Vincan E, Darcy P K, Smyth M J, et al. Frizzled-7receptor    ectodomain expression in a colon cancer cell line induces    morphological change and attenuates tumor growth. Differentiation    73:142-53(2005).-   34. Zi X, Guo Y, Simoneau A R, et al. Expression of Frzb/secreted    Frizzled-related protein 3, a secreted Wnt antagonist, in human    androgen-independent prostate cancer PC-3 cells suppresses tumor    growth and cellular invasiveness. Cancer Res 65:9762-70(2005).-   35. Dennis S, Aikawa M, Szeto W, d'Amore P A, Papkoff J. A secreted    frizzled related protein, FrzA, selectively associates with Wnt-1    protein and regulates wnt-1 signaling. J Cell Sci 112(Pt    21):3815-3820(1999).-   36. Lu D, Zhao Y, Tawatao R, et al. Activation of the Wnt signaling    pathway in chronic lymphocytic leukemia. Proc Natl Acad Sci USA    101:3118-23(2004).-   37. Franke T F, Hornik C P, Segev L, Shostak G A, Sugimoto C.    PI3K/Akt and apoptosis: size matters. Oncogene 22:8983-8998(2003).-   38. Kurihara T, Brough D E, Kovesdi I, Kufe D W. Selectivity of a    replication-competent adenovirus for human breast carcinoma cells    expressing the MUC1 antigen. J Clin Invest 106:763-771(2000).-   39. Zhou Z, Zhou R R, Guan H, Bucana C D, Kleinerman E S. E1A gene    therapy inhibits angiogenesis in a Ewing's sarcoma animal model. Mol    Cancer Ther 2:1313-9(2003).-   40. Saito Y, Sunamura M, Motoi F, et al. Oncolytic    replication-competent adenovirus suppresses tumor angiogenesis    through preserved E1A region. Cancer Gene Ther 13:242-52(2006).-   41. Sato H, Kida Y, Mai M, et al. Expression of genes encoding type    IV collagen-degrading metalloproteinases and tissue inhibitors of    metalloproteinases in various human tumor cells. Oncogene    7:77-83(1992).

What is claimed is:
 1. A method for treating cancer selected from thegroup consisting of stomach cancer, lung cancer, breast cancer, ovariancancer, liver cancer, brain cancer, prostate cancer, sarcoma,bronchogenic cancer, nasopharyngeal cancer, laryngeal cancer, pancreaticcancer, bladder cancer, colon cancer, and uterine cervical cancer, themethod comprising: administering to a subject in need thereof apharmaceutically effective amount of a composition comprising apolypeptide having the amino acid sequence of SEQ ID NO:2.
 2. The methodaccording to claim 1, wherein the composition inhibits activation of Wntsignaling pathway in a cancer cell.
 3. The method according to claim 2,wherein the composition inhibits activation of Wnt signaling pathway ina cancer cell through binding of the polypeptide having the amino acidsequence of SEQ ID NO:2 to Wnt 3a protein.